Fibroblast growth factor receptor activating gene 1 and related compositions and methods

ABSTRACT

A novel gene designated as FRAG1 from and its encoded protein is disclosed. A fusion protein called FGFR2-ROS, which is formed by chromosomal rearrangement of rat FRAG1 with FGFR2 is also disclosed. Methods of producing FRAG1 protein, related fusion proteins, and antibodies against FRAG1 are disclosed, as are related pharmaceuticals and methods of using such nucleic acids, polypeptides, and antibodies are also disclosed.

CROSS-REFERENCE TO RELATED CASES

This is a divisional of U.S. patent application Ser. No. 10/461,180filed Jun. 12, 2003 now U.S. Pat. No. 6,831,170, which is a divisionalof U.S. patent application Ser. No. 09/942,858 filed Aug. 29, 2001, nowU.S. Pat. No. 6,608,181, which is a divisional application of U.S.patent application Ser. No. 09/202,548, filed Dec. 15, 1998, now U.S.Pat. No. 6,323,316, which is a §371 application of PCT/US97/10660, filedJun. 18, 1997, which claims the benefit of U.S. Provisional ApplicationNo. 60/020,009, filed Jun. 18, 1996, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates to a novel gene that activates Fibroblast GrowthFactor Receptor 2 (FGFR2).

The fibroblast growth factors (FGFs) are a family of related proteinswith roles in mitogenesis, differentiation, wound healing, andorganogenesis (Basilico et al. Adv. Cancer Res. 59:115-165, 1992). Thebiological responses of FGFs are mediated through specific high-affinityreceptor tyrosine kinases (Givol and Yayon, FASEB J. 6:3362-3369, 1992).Four distinct classes of fibroblast growth factor receptor (FGFR) thatencode structurally related proteins have been identified: FGFR1/F1g,FGFR2/Bek, FGFR3, and FGFR4 (Dionne et al., EMBO J. 9:2685-2692, 1990);Miki et al., Science 251:72-75, 1991; Keegan et al., Proc. Natl. Acad.Sci. USA 88:1095-1099, 1991; and Partanen et al., EMBO J. 10:1347-1354,1992).

Activating mutations (i.e. mutations, leading to highly phosphorylatedreceptors, that are associated with various cancer states) intransmembrane domains of EGF receptor and FGFR3 were reported in ratleukemia and human achondroplasia, respectively (Ben-Levy et al., J.Biol. Chem. 267:17304-17313, 1992; and Shiang et al., Cell, 78:335-342,1994). Furthermore, several growth factor receptors have been found tobe activated by chromosomal rearrangement in cancer cells (Sawyers etal., Cell 77:171-173, 1994), including EGFR in leukemias, NGFR (TRK) incolon and thyroid carcinomas, HGFR (MET) in gastric carcinomas, RET inthyroid carcinomas, ALK in lymphomas, and PDGFRβ in myeloid leukemias.

One chromosomal rearrangement associated with chronic myelomonocyticleukemia was shown to create an expressed fusion between a novel genesequence, tel, and the tyrosine kinase domain of the growth factorreceptor PDGFRβ (Golub et al., Cell 77:307-316, 1994). tel appears to bea member of the ets gene family, members of which encode transcriptionfactors. Genes other than growth factor receptors (GFRs), are activatedand cause tumorigenesis by gene fusions resulting from chromosomalarrangement. For example, a majority of the cases of chronic myelomaleukemia (CML) contain a chromosomal rearrangement between chromosome 9and 22 (t(9;22)(q34;q11) that results in a fusion of the oncogene α-abl,a cytoplasmic kinase, to bcr, which promotes myeloid tumorigenesis andis used as a diagnostic for the malignancy (Bernards et al., Mol. CellBiol. 7:3231-3236, 1987; and Konopka et al., Proc. Nat'l. Acad. Sci.82:1810-1813, 1985).

SUMMARY OF THE INVENTION

We have cloned and sequenced rat and human FRAG1 cDNAs (SEQ ID NO: 5 and11, respectively). Chromosomal rearrangements resulting in a fusion ofFRAG1 to fibroblast growth factor receptor 1 (FGFR2) causes a potentactivation of FGFR2. Based on these discoveries, the present inventionprovides compositions and methods related to the isolated rat and humanFRAG1 genes.

Accordingly, one embodiment of the invention is an isolated nucleic acidthat comprises: (a) a sequence of at least 15 contiguous nucleotides ofa native FRAG1 nucleic acid or the complement thereof; or (b) a sequenceof at least 100 nucleotides having at least 70%, more preferably atleast 80%, and yet more preferably at least 90%, and most preferably atleast 95% nucleotide sequence similarity with a native FRAG1 nucleicacid (or a complement thereof), particularly with the rat or human FRAG1nucleic acid (SEQ ID NO: 5 and 11, respectively). According to anotherembodiment, the isolated nucleic acid encodes a polypeptide sequencehaving only silent or conservative substitutions to a native rat orhuman FRAG1 polypeptide. According to another embodiment, the isolatednucleic acid encodes a native or wild-type rat or human FRAG1polypeptide (SEQ ID NO: 6 and 12, respectively). According to anotherembodiment, the isolated nucleic acid encodes a polypeptide that, whenexpressed as an in-frame fusion with FGFR2, stimulates the transformingactivity and autophosphorylation of FGFR2.

Another embodiment of the invention is a cell that comprises such FRAG1nucleic acids, including expression vectors that are suitable forexpression of recombinant FRAG1 polypeptides in a host cell. Such cellscan be used to making FRAG1 polypeptides by culturing the cells underconditions suitable for expression of the FRAG1 polypeptide, followed byisolation of the expressed FRAG1 polypeptide from the cell byconventional methods.

FRAG1 nucleic acids are useful for detecting an abnormality in achromosome (e.g., a chromosomal rearrangement such as a fusion of FRAG1to another gene that, when expressed, produces a fusion polypeptide) ofa subject comprising the steps of: incubating chromosomes of a subjectthat comprises a chromosomal abnormality (e.g., a rearrangementresulting in a fusion of FRAG1 to another gene) with a FRAG1 nucleicacid probe or primer under conditions that cause the probe or primer tohybridize specifically with a native FRAG1 sequence. Hybridization ofthe probe or primer to the subject's chromosomes is compared withhybridization to a normal control chromosome, i.e., a chromosome knownto lack the abnormality, thereby allowing the abnormality to bedetected. Detection of chromosomal abnormalities can be accomplished,for example, by fluorescence in situ hybridization or nucleic acidamplification techniques. Such chromosome abnormalities may bediagnostic for disease states such as a neoplasia (e.g., anosteosarcoma).

Another embodiment of the invention is isolated FRAG1 polypeptides,e.g., polypeptides encoded by a FRAG1 nucleic acid as described above.For example, FRAG1 polypeptides according to various embodiments of theinvention include polypeptides that comprise at least 10 consecutiveamino acids of a native rat or human FRAG1 polypeptide; polypeptideshaving at least 70% amino acid sequence homology to a native rat orhuman FRAG1 polypeptide; and full-length native FRAG1 polypeptides.

Another embodiment of the invention is an antibody that is specific fora native FRAG1 polypeptide.

FRAG1 genes can be isolated from species other than rat or human bycontacting nucleic acids of the species with a FRAG1 probe or primerunder at least moderately stringent nucleic acid hybridizationconditions and isolating the FRAG1 gene to which the probe or primerhybridizes. For example, a cDNA or genomic library of the species can bescreened with a FRAG1 probe, or primer, or mRNA or genomic DNA can besubjected to a nucleic acid amplification procedure to amplify a FRAG1homolog of the species. In an alternative method of obtaining a FRAG1gene of a species other than rat or human, an expression librarycomprising a plurality of cells that each express a recombinantpolypeptide is contacted with a FRAG1-specific antibody under conditionsthat cause the FRAG1-specific antibody to specifically bind to arecombinant polypeptide encoded by a FRAG1 gene, thereby identifying acell that expresses the FRAG1 gene. The FRAG1 gene can then be isolatedfrom the cell that expresses the FRAG1 gene.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of FGFR2 isoforms and a C-terminaldomain comparison of FGFR2-ROS with other FGFR2 cDNAs. The location ofsignal peptide (S), acidic (A), immunoglobulin-like (IG2, IG3),transmembrane (TM), juxtamembrane (JM), kinase insert (KI), tyrosinekinase (TK1, TK2) and carboxy-terminal (CT) regions are shown above theschematic. KGFR-type isoforms contain the sequence encoded by the K exonof the FGFR2/BEK gene in the second half of IG3 domain (shaded area).The variant C-terminal domain of FGFR2-ROS is indicated by a dashed box.All of these cDNAs are similar unless otherwise indicated and areinserted in a eukaryotic expression vector pCEV27 (Miki et al., Gene83:137-146, 1989).

FIG. 2 shows the deduced C-terminal amino acid sequences (beginning atposition 759) of FGFR2 cDNAs (FGFR2-WT, SEQ ID NO: 1; FGFR2-ET, SEQ IDNO: 2; FGFR2-ROS, SEQ ID NO: 3). The amino acid sequence and numberingof wild-type rat FGFR2 (rat FGFR2-WT, SEQ ID NO: 1) is according to athree Ig-domain form of FGFR2-WT (Takagi et al., J. Biol. Chem.269:23743-23749, 1994). The extended C-terminal domain of FGFR2-ROS (SEQID NO: 3) is depicted by a series of dots. Numbers above the sequencedenote the position of C-terminal tyrosine residues and asterisksrepresent termination codons.

FIG. 3 shows a comparison of FGFR2-ROS and rat FRAG1 cDNAs. Thepredicted amino acid sequence for FGFR2 (starting at position 759) andrat FRAG1 (underlined) at the fusion junction is shown above theschematic. A vertical line indicates the breakpoint. The first ATG inthe open reading frame of rat FRAG1 is shown by an arrowhead. Theportions of FGFR2-ROS derived from rat FRAG1 are indicated by hatchedboxes. Untranslated regions are depicted by solid lines.

FIG. 4 shows the nucleotide and predicted amino acid sequences of ratFRAG1 (SEQ ID NO: 5 and 6, respectively). The breakpoint in rat FRAG1(between G and C, underlined) occurs 5′ to the ATG (boxed) used fornumbering the predicted amino acid sequence. The entire open readingframe was translated from the first amino acid sequence, but the aminoacids are numbered (right) from the predicted start codon (boxed). Atermination codon is marked by an asterisk.

FIG. 5 shows an alignment of the 5′ ends of rat FRAG1 and correspondingsequences in a 30 kDa nematode gene (TO4A8.12) (SEQ ID NO: 7) and a107.9 kDa yeast protein (SC10 kD) (SEQ ID NO: 8). A particularlywell-conserved region is bracketed.

FIG. 6 shows an alignment of the deduced polypeptide sequences of ratFRAG1 (SEQ ID NO: 6), TO4A8.12 (SEQ ID NO: 9), and SC108 kD (SEQ ID NO:10).

FIG. 7 shows a nucleotide sequence of a full-length human FRAG1 cDNA(FRAG1-19) (SEQ ID NO: 11) and the amino acid sequence thereof (SEQ IDNO: 12).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a novel gene, FRAG1, which has beenisolated from rat (see Lorenzi et al., Proc. Natl. Acad. Sci. USA93:8956-8961, 1996, which is incorporated herein by reference) andhuman.

The rat FRAG1 cDNA was isolated as follows as a result of screening anosteosarcoma (ROS) cDNA library that was transformed into NIH 3T3 cellsand transformants. One clone was shown to encode an isoform of FGFR2,FGFR2-ROS. It was determined that FGFR2-ROS was created by chromosomalrearrangement and included a C-terminal stretch of 313 amino acidsoriginating from a novel gene, FRAG1. A probe derived from the FRAG1portion of the FGFR2-ROS cDNA was used to isolate a gene that in ratencodes a protein of 254 amino acids long (SEQ ID NO: 6).

The human FRAG1 cDNA was isolated by screening a library prepared fromhuman fibroblast cells with a rat FRAG1 cDNA probe. Internal DNAsequence revealed that these cDNAs encoded the human homolog of ratFRAG1. The sequence of approximately 90% of the human FRAG1 cDNA hasbeen determined (FIG. 7 and SEQ ID NO: 10). A search of nucleotidesequence databases revealed that human FRAG1 is highly related to therat FRAG1 sequence but contains unique regions not present in the ratFRAG1 sequence. The size of the human FRAG1 mRNA on Northern blotssuggest that the human FRAG1-5CA clone is full length.

FGFR2-ROS was shown to be phosphorylated to a much greater extent thanother FGFR2 isoforms and to be fully activated in a ligand-independentmanner. The greater phosphorylation and highly activated state ofFGFR2-ROS were shown to depend upon the C-terminal FRAG1 sequence. Itwas determined that FGFR2-ROS forms unusually stable dimers.Constitutive dimerization and autophosphorylation may underlie thepotent transforming activity of FGFR2-ROS. It was also found that FRAG1protein tends to accumulate in the Golgi complex. Thus, the alteredsubcellular localization of FGFR2-ROS could also explain its highlyactivated state.

FGFR2-FRAG1 fusions are a likely cause of osteosarcoma. Other diseasestates are known to involve chromosomal rearrangement. In particular,several growth factor receptors have been found to be activated bychromosomal rearrangement in cancer cells (Sawyers and Denny, Cell77:171-173, 1994). Thus, the FRAG1 gene may be involved in otherchromosomal rearrangements that are responsible for or associated withother disease states.

Definitions and Methods

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991, and Lewin,Genes V, Oxford University Press: New York, 1994. The standardnomenclature for DNA bases (see 37 CFR § 1.822) and the standard one-and three-letter nomenclature for amino acid residues are used.

Nucleic Acids

“FRAG1 Gene”. The term “FRAG1 gene” or “FRAG1” refers to a nativeFRAG1-encoding nucleic acid or polynucleotide or a fragment thereof,e.g., the native FRAG1 cDNA or genomic sequences and alleles andhomologs thereof. The term also encompasses variant forms of a nativeFRAG1 nucleic acid sequence or fragment thereof as discussed below,preferably a nucleic acid that encodes a polypeptide having FRAG1biological activity. Native FRAG1 sequences include cDNA sequences andthe corresponding genomic sequences (including flanking or internalsequences operably linked thereto, including regulatory elements and/orintron sequences).

Both double-stranded and single-stranded FRAG1 nucleic acids areencompassed. With reference to single-stranded forms of FRAG1 nucleicacids, “complementary nucleic acids” or “complements” (i.e., nucleicacids that base pair with such single-stranded forms) are alsoencompassed.

“Native”. The term “native” refers to a naturally-occurring(“wild-type”) nucleic acid or polypeptide, including, but not limitedto, the native rat or human FRAG1 nucleic acid or the polypeptideencoded thereby.

“Homolog”. A “homolog” of a FRAG1 gene is a gene sequence encoding aFRAG1 polypeptide isolated from a species other than a reference FRAG1gene. For example, with reference to rat FRAG1, the human FRAG1 genedisclosed herein is a homolog. FRAG1 homologs from a variety of speciesother than rat and human can be readily obtained using probes andprimers derived from rat and/or human FRAG1.

“Isolated”. An “isolated” nucleic acid is one that has beensubstantially separated or purified away from other nucleic acidsequences in the cell of the organism in which the nucleic acidnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, by conventional nucleic acid-purification methods. The term alsoembraces recombinant nucleic acids and chemically synthesized nucleicacids.

Fragments, Probes, and Primers. A fragment of a FRAG1 nucleic acid is aportion of a FRAG1 nucleic acid that is less than full-length andcomprises at least a minimum length capable of hybridizing specificallywith a native FRAG1 nucleic acid under stringent hybridizationconditions. The length of such a fragment is preferably at least 15nucleotides, more preferably at least 20 nucleolides, yet morepreferably at least 30 nucleotides, and most preferably at least 50nucleotides of a native FRAG1 nucleic acid sequence.

Nucleic acid probes and primers can be prepared based on a native FRAG1gene sequence. A “probe” is an isolated nucleic acid to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, or enzyme.“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase. e.g., a DNA polymerase. Primerpairs can be used for amplification of a nucleic acid sequence, e.g., bythe polymerase chain reaction (PCR) or other conventional nucleic-acidamplification methods.

Probes and primers are generally 15 nucleotides or more in length,preferably 20 nucleotides or more, more preferably 25 nucleotides, andmost preferably 30 nucleotides or more. Such probes and primershybridize specifically to the FRAG1 sequence under high stringencyhybridization conditions and hybridize specifically to a native FRAG1sequence of another species under at least moderately stringentconditions. Preferably, probes and primers according to the presentinvention have complete sequence similarity with the native FRAG1sequence, although probes differing from the FRAG1 sequence and thatretain the ability to hybridize to native FRAG1 sequences may bedesigned by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992); and Innis et al., PCR Protocols: AGuide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the native FRAG1 sequences disclosed hereincan be used to confirm (and, if necessary, to correct) the disclosed ratand human FRAG1 sequences by conventional methods, e.g., by simplyresequencing the deposited human FRAG1 cDNA or by recloning andsequencing a FRAG1 cDNA or genomic sequence by conventional methods.

Substantial Similarity. A first nucleic acid is “substantially similar”to a second nucleic acid if, when optimally aligned (with appropriatenucleotide insertions or deletions) with the other nucleic acid (or itscomplementary strand), there is at least about 70% nucleotide sequenceidentity, preferably at least about 80% identity, and most preferably atleast about 90% identity. Sequence similarity can be determined bycomparing the nucleotide sequences of two nucleic acids using sequenceanalysis software such as the Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,Madison, Wis.

Alternatively, two nucleic acids are substantially similar if theyhybridize under stringent conditions, as defined below.

“Operably Linked”. A first nucleic-acid sequence is “operably” linkedwith a second nucleic-acid sequence when the first nucleic-acid sequenceis placed in a functional relationship with the second nucleic-acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in readingframe.

“Recombinant”. A “recombinant” nucleic acid is made by an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

Techniques for nucleic-acid manipulation are well-known (see. e.g.,Sambrook et al., 1989, and Ausubel et al., 1992). Methods for chemicalsynthesis of nucleic acids are discussed, for example, in Beaucage andCarruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J.Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids canbe performed, for example, on commercial automated oligonucleotidesynthesizers.

Vectors, Transformation, Host cells. Natural or synthetic nucleic acidsaccording to the present invention can be incorporated into recombinantnucleic-acid constructs or vectors that can be introduced into andreplicated in a host cell. Such vectors include selectable or screenablemarker genes, well-known sequences that allow the vector to bereplicated in a given prokaryotic or eukaryotic host cell (e.g., originof replication), transcriptional and translational control sequencesthat are functional in the host cell (e.g., promoters, enhancers),secretion signals, etc. Compositions and methods for preparing and usingvectors and host cells, including host cell transformation, arediscussed, inter alia, in Sambrook, 1989, or Ausubel, 1994. Mammalian orother eukaryotic host cells, such as those of yeast, filamentous fungi,plant, insect, amphibian or avian species, are useful for production ofthe proteins of the present invention by conventional methods.

A cell, tissue, organ, or organism into which a foreign nucleic acid,such as a recombinant vector, has been introduced is considered“transformed”, “transfected”, or “transgenic.”

Nucleic acid constructs can be introduced into a host cell by anysuitable conventional method, including electroporation; transfectionemploying calcium chloride, rubidium chloride calcium phosphate,DEAE-dextran, or other substances; microprojectile bombardment;lipofection; infection (where the vector is an infectious agent, such asa retroviral genome); etc. See, e.g., Sambrook, 1989, and Ausubel, 1994.

Nucleic-Acid Hybridization; “Stringent Conditions”; “Specific”. Thenucleic-acid probes and primers of the present invention hybridize understringent conditions to a target DNA sequence, e.g., to a FRAG1 gene.

The term “stringent conditions” is functionally defined with regard tothe hybridization of a nucleic-acid probe to a target nucleic acid(i.e., to a particular nucleic-acid sequence of interest) by thespecific hybridization procedure discussed in Sambrook et al., 1989, at9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58;Kanehisa, Nucl. Acids Res. 12:203-213, 1984; and Wetmur and Davidson, J.Mol. Biol. 31:349-370, 1968. “Moderate stringency” hybridizationconditions are defined as hybridization at 60° C. in a hybridizationsolution including 6×SSC, 5× Denhardt's reagent, 0.5% SDS, 100 μg/mLdenatured, fragmented salmon sperm DNA, and a labeled probe (Sambrook,1989), and “high stringency” conditions are hybridization at 65° C., orpreferably at 68° C., in the same hybridization solution.

Regarding the amplification of a target nucleic-acid sequence (e.g., byPCR) using a particular amplification primer pair, “high stringencyconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under given hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

Nucleic-Acid Amplification. As used herein, “amplified DNA” refers tothe product of nucleic-acid amplification of a target nucleic-acidsequence. Nucleic-acid amplification can be accomplished by any of thevarious nucleic-acid amplification methods known in the art, includingthe polymerase chain reaction (PCR). A variety of amplification methodsare known in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990.

Nucleotide-Sequence Variants of Native FRAG1 Nucleic Acids and AminoAcid Sequence Variants of Native FRAG1 Proteins. Using the nucleotideand the amino-acid sequence of the FRAG1 polypeptides disclosed herein,those skilled in the art can create DNA molecules and polypeptides thathave minor variations in their nucleotide or amino acid sequence.

“Variant” DNA molecules are DNA molecules containing minor changes in anative FRAG1 sequence, i.e., changes in which one or more nucleotides ofa native FRAG1 sequence is deleted, added, and/or substituted,preferably while substantially maintaining a FRAG1 biological activity.Variant DNA molecules can be produced, for example, by standard DNAmutagenesis techniques or by chemically synthesizing the variant DNAmolecule or a portion thereof. Such variants preferably do not changethe reading frame of the protein-coding region of the nucleic acid andpreferably encode a protein having no change, only a minor reduction, oran increase in FRAG1 biological function.

Amino-acid substitutions are preferably substitutions of singleamino-acid residues. DNA insertions are preferably of about 1 to 10contiguous nucleotides and deletions are preferably of about 1 to 30contiguous nucleotides. Insertions and deletions are preferablyinsertions or deletions from an end of the protein-coding or non-codingsequence and are preferably made in adjacent base pairs. Substitutions,deletions, insertions or any combination thereof can be combined toarrive at a final construct.

Preferably, variant nucleic acids according to the present invention are“silent” or “conservative” variants. “Silent” variants are variants of anative FRAG1 sequence or a homolog thereof in which there has been asubstitution of one or more base pairs but no change in the amino-acidsequence of the polypeptide encoded by the sequence. “Conservative”variants are variants of the native FRAG1 sequence or a homolog thereofin which at least one codon in the protein-coding region of the gene hasbeen changed, resulting in a conservative change in one or more aminoacid residues of the polypeptide encoded by the nucleic-acid sequence,i.e., an amino acid substitution. A number of conservative amino acidsubstitutions are listed below. In addition, one or more codons encodingcysteine residues can be substituted for, resulting in a loss of acysteine residue and affecting disulfide linkages in the FRAG1polypeptide.

Substantial changes in function are made by selecting substitutions thatare less conservative than those listed in Table 1, e.g., causingchanges in: (a) the structure of the polypeptide backbone in the area ofthe substitution; (b) the charge or hydrophobicity of the polypeptide atthe target site; or (c) the bulk of an amino acid side chain.Substitutions generally expected to produce the greatest changes inprotein properties are those in which: (a) a hydrophilic residue, e.g.,seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteineor proline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

TABLE 1 Conservative Amino Acid Substitutions Original ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp,Phe Val Ile, Leu

Deposit Information. The human FRAG1 cDNA has been deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas. Va. 20110-2209 under terms of the Budapest Treaty on Jun. 9,1997, under ATCC No. 209102.

“FRAG1 Protein”. The term “FRAG1 protein” (or polypeptide) refers to aprotein encoded by a FRAG1 nucleic acid, including alleles, homologs,and variants of a native FRAG1 nucleic acid, for example. A FRAG1polypeptide can be produced by the expression of a recombinant FRAG1nucleic acid or be chemically synthesized. Techniques for chemicalsynthesis of polypeptides are described, for example, in Merrifield, J.Amer. Chem. Soc. 85:2149-2156, 1963.

Polypeptide Sequence Homology. Ordinarily, FRAG1 polypeptidesencompassed by the present invention are at least about 70% homologousto a native FRAG1 polypeptide, preferably at least about 80% homologous,and more preferably at least about 95% homologous. Such homology isconsidered to be “substantial homology,” although more important thanshared amino-acid sequence homology can be the common possession ofcharacteristic structural features and the retention of FRAG1 biologicalactivity.

Polypeptide homology can be analyzed by any conventional method, e.g.,by using sequence analysis software such as the Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, Madison, Wis.) or other well-known software.

“Isolated,” “Purified,” “Homogeneous.” A polypeptide is “isolated” if ithas been separated from the cellular components (nucleic acids, lipids,carbohydrates, and other polypeptides) that naturally accompany it. Sucha polypeptide can also be referred to as “pure” or “homogeneous” or“substantially” pure or homogeneous. Thus, a polypeptide which ischemically synthesized or recombinant (i.e., the product of theexpression of a recombinant nucleic acid, even if expressed in ahomologous cell type) is considered to be isolated. A monomericpolypeptide is isolated when at least 60% by weight of a sample iscomposed of the polypeptide, preferably 90% or more, more preferably 95%or more, and most preferably more than 99%. Protein purity orhomogeneity is indicated, for example, by polyacrylamide gelelectrophoresis of a protein sample, followed by visualization of asingle polypeptide band upon staining the polyacrylamide gel; highpressure liquid chromatography; or other conventional methods.

Protein Purification. The polypeptides of the present invention can bepurified by any of the means known in the art. Various methods ofprotein purification are described, e.g., in Guide to ProteinPurification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, SanDiego, 1990; and Scopes, Protein Purification: Principles and Practice,Springer Verlag, New York, 1982.

Variant and Modified Forms of FRAG1 Polypeptides. Encompassed by theFRAG1 polypeptides of the present invention are variant polypeptides inwhich there have been substitutions, deletions, insertions or othermodifications of a native FRAG1 polypeptide. The variants substantiallyretain structural characteristics and biological activities of acorresponding native FRAG1 polypeptide and are preferably silent orconservative substitutions of one or a small number of contiguous aminoacid residues.

A native FRAG1 polypeptide sequence can be modified by conventionalmethods, e.g., by acetylation, carboxylation, phosphorylation,glycosylation, ubiquitination, and labeling, whether accomplished by invivo or in vitro enzymatic treatment of a FRAG1 polypeptide or by thesynthesis of a FRAG1 polypeptide using modified amino acids.

Labeling. There are a variety of conventional methods and reagents forlabeling polypeptides and fragments thereof. Typical labels includeradioactive isotopes, ligands or ligand receptors, fluorophores,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al., 1989 and Ausubel et al., 1992.

Polypeptide Fragments. The present invention also encompasses fragmentsof a FRAG1 polypeptide that lack at least one residue of a nativefull-length FRAG1 polypeptide. Preferably, such a fragment retains FRAG1biological activity or possesses a characteristic functional domain oran immunological determinant characteristic of a native FRAG1polypeptide. Immunologically active fragments typically have a minimumsize of 7 to 17 or more amino acids. Preferred embodiments of thepolypeptides of the invention include at least 10, more preferably atleast 15, yet more preferably at least 20, and most preferably at least25 consecutive amino acids of a native FRAG1 polypeptide.

The terms “biological activity”, “biologically active”; “activity” and“active” refer primarily to the characteristic biological activity oractivities of a native FRAG1 polypeptide, including, but not limited to,the ability to stimulate the transforming activity andautophosphorylation of FGFR2 when fused thereto, as described in greaterdetail in Example 1.

Fusion Polypeptides. The present invention also provides fusionpolypeptides including, for example, heterologous fusion polypeptides inwhich a FRAG1 polypeptide sequence is joined to a fusion partner. Suchfusion polypeptides can exhibit biological properties (such as substrateor ligand binding, enzymatic activity, antigenic determinants, etc.)derived from each of the fused sequences. Fusion polypeptides arepreferably made by the expression of recombinant nucleic acids producedby standard techniques. Fusion of FRAG1 to FGFR2 has been found topotently activate FGFR2. It is expected that fusion of FRAG1 to othergrowth factors, among other protein fusion partners, also will stimulatetransforming activity and/or autophosphorylation of the fusion partner.

Polypeptide Sequence Determination. The sequence of a polypeptide of thepresent invention can be determined by any of various methods known inthe art.

Antibodies

The present invention also encompasses polyclonal and/or monoclonalantibodies capable of specifically binding to a FRAG1 polypeptide and/orfragments thereof. Such antibodies are raised against a FRAG1polypeptide or fragment thereof and are capable of distinguishing aFRAG1 polypeptide from other polypeptides, i.e., are “FRAG1-specific.”Encompassed by the present invention are double- and single-chainFRAG1-binding antibody fragments (e.g., Fv, Fab, (Fab′)₂, etc.),chimeric antibodies, humanized antibodies, and other modified antibodiesmade by conventional methods. Conventional methods are used for thepreparation and use of antibodies according to the present invention,including various immunoassay techniques and applications, see, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, 2d ed, AcademicPress, New York, 1986; and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988.FRAG1-specific antibodies are useful, for example in: purifying a FRAG1polypeptide from a biological sample, such as a host cell expressingrecombinant a FRAG1 polypeptide; in cloning a FRAG1 allele or homologfrom an expression library; as antibody probes for protein blots andimmunoassays; etc.

Anti-FRAG1 antibodies can be labeled by any of a variety of conventionalmethods. Suitable labels include, but are not limited to, radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent agents,chemiluminescent agents, magnetic particles, etc.

Uses of Rat and Human FRAG1 Nucleic Acids and Polypeptides

Obtaining Alleles and Homologs of Rat or Human FRAG1. Alleles andhomologs of rat and human FRAG1 can readily be obtained from otherspecies using probes and primers derived from the sequences disclosedherein by conventional methods, e.g., by screening a cDNA or genomiclibrary with a probe that specifically hybridizes to a native FRAG1sequence under at least moderately stringent conditions (e.g., to therat FRAG1 cDNA or a fragment thereof), by PCR or another amplificationmethod using a primer or primers that specifically hybridize to a nativeFRAG1 sequence under at least moderately stringent conditions, or byidentification of FRAG1 alleles or homologs in an expression libraryusing FRAG1-specific antibodies.

Probes and primers based on the rat FRAG1 sequence disclosed herein canalso be used to obtain genes having substantial sequence homology withrat FRAG1 but different biological activity.

Diagnostics. Most tumor types undergo chromosomal rearrangements thatmay be consistently associated with and diagnostic of particular tumortypes. The sites of these rearrangements often map to genes that arethought to be critically involved in tumor progression. For example,several genes involved in cellular growth control are known to undergorearrangement with other genes in tumor cells. In chronic myelomonocyticleukemia (CMML), a chromosomal rearrangement between the genes encodingplatelet-derived growth factor receptor (PDGFR) and tel results in theexpression of a PDGFR-tel fusion protein in the tumor cells and isdiagnostic for CML. Several receptor tyrosine kinase receptors undergostructural alterations or are overexpressed in human cancer. In the caseof Tpr, an unknown gene, chromosomal rearrangements at the Tpr locusfrequently occur with multiple receptors, thereby converting silentreceptors into potent oncogenes.

We have isolated a constitutively active form of FGFR2 from cellsderived from an osteosarcoma that is activated by chromosomalrearrangement with FRAG1. The FGFR2 gene has been shown to be involvedin cancers of the stomach and in diseases of bone malformation. Manyother growth factor receptors, including FGFR1, FGFR3, EGFR, erbB2, anderbB3 have been implicated in human disease. FRAG1 rearrangements withthe genes encoding these receptors may be utilized as a marker forcancers such as osteosarcoma, for example, and other disease states.Such rearrangements may be detected by using FRAG1-derived primers fornucleic acid amplification (e.g., PCR) or FRAG1-derived probes forfluorescence in situ hybridization (FISH), for example.

Fluorescence in situ hybridization (FISH), for example, is one usefultechnique for identifying and characterizing chromosomal abnormalitiesby staining specific chromosomes in a manner that allows numerical andstructural aberrations to be easily evaluated in either metaphase orinterphase cells (Pinkel et al., Proc. Natl. Acad. Sci. USA85:9138-9142, 1988). The characterization of cytogenetic aberrations intumors has contributed to the understanding of carcinogenesis, tumorprogression, and to clinical management decisions. A “marker chromosome”is a chromosome differing from a normal chromosome, for example, in sizeand/or banding pattern, that is characteristic of the cells in aparticular tumor or other disease state and is not found in normalcells. Such marker chromosomes frequently result from the rearrangementand recombination of portions of a normal chromosome or several normalchromosomes. The availability of rat and human FRAG1 genes is permitsthe identification of marker chromosomes that are diagnostic forosteosarcoma and other disease states, particularly neoplasias.

Therapeutic uses. Agents capable of inhibiting FRAG1 activity, such asFRAG1-specific antibodies, FRAG1-derived antisense or triplexhelix-forming nucleic acids, or other agents, are useful for treatingdiseases involving FRAG1 rearrangements by blocking FRAG1-mediatedactivation of receptors.

Drug Screening. FRAG1 polypeptides are useful for screening compounds byconventional drug-screening methodologies. For example, compounds thatbind to FRAG1 polypeptides can be identified by any of variouswell-known competitive binding assays, including methods for screeningcombinatorial libraries in which large numbers of different peptides aresynthesized on a solid substrate or presented by a recombinantexpression as part of a phage protein (e.g., a coat protein of afilamentous phage), for example. The peptide test compounds are reactedwith a FRAG1 polypeptide and washed. Bound FRAG1 polypeptide is thendetected to determine the location of the bound peptide, the sequence ofwhich can be determined readily.

The invention will be better understood by reference to the followingExamples, which are intended to merely illustrate the best mode nowknown for practicing the invention. The scope of the invention is not tobe considered limited thereto.

EXAMPLES Example 1 Cloning and Analysis of Rat FRAG1

Materials and Methods

FGFR2 expression constructs and transformants. FGFR2-WT was engineeredby replacing the variant carboxyl-terminal domain of p822 with thenormal C-terminus of the KGFR at the BglII site (amino acid position696). The coding regions of mouse and rat KGFR are identical in thisregion. A termination codon was inserted by PCR to introduce a deletionin the C-terminus of FGFR2-WT at position 764 to generate Δ764-.Expression constructs were transfected into NIH 3T3 fibroblasts bycalcium-phosphate precipitation (Wigler et al., Cell 14:725-731, 1978).Mass cultures of NIH 3T3 cells expressing the recombinant proteins wereobtained by selection with G418 (750 μg/ml).

Protein Analysis. Immunoprecipitation and immunoblot analysis usingIgG-purified rabbit polyclonal antiserum generated against the aminoacids 476-822 of the cytoplasmic domain of FGFR2 (αFGFR2, 1:1000) oranti-phosphotyrosine monoclonal antibody (αPTYR, 1:1000, UpstateBiotechnology, Inc.) was conducted as described (Miki et al., Science251:72-75, 1991). For covalent crosslinking experiments, cells wereincubated in 0.3 mM disuccinimidyl suberate dissolved indimethylsulfoxide (DMSO) or DMSO alone for 20 minutes at 4° C. Thecrosslinking reaction was terminated by addition of 10 mM Tris-HCl (pH7.5), 200 mM glycine, 2 mM EDTA, and lysate were prepared and analyzedas described above.

Immunofluorescent staining. pCEV29F3, an expression vector encodingthree tandem copies of the FLAG nonapeptide was engineered by amodification of pCEV27. Triple FLAG epitope-tagged rat FRAG1 (F3-FRAG1)was generated by inserting rat FRAG1 cDNA (encoding amino acids 1-244)into pCEV29F3 in frame with the triple FLAG epitope. NIH 3T3transfectants of F3-FRAG1 or pCEV29F3 were subjected toimmunofluorescence staining using an anti-FLAG monoclonal antibody(1:200, IBI) as described (Jackman et al., EMBO J. 14:1646-1654, 1995).Bound antibody was visualized using a lissamine rhodamine(LRSC)-conjugated goat anti-mouse antisera (1:200, Jackson Laboratories,Bar Harbor, Me.).

Results

Expression cloning of a transforming FGFR2 from osteosarcoma cells. Anovel oncogene, ost, was previously isolated from ROS 17/2.8 cells usingan expression cloning system (Miki et al., Nature 362:462-465, 1993).However, the ost oncogene showed no evidence of rearrangement in ROScells, indicating that it was not responsible for osteosarcomaformation. Several more plasmids with transforming activity were rescuedfrom the foci induced by the ROS cell expression cDNA library. Sequenceanalysis of the 5′-end of the cDNA inserts revealed that one of theseplasmids, p822, encodes a fibroblast growth factor, FGFR2. p822 showedhigh-titered transforming activity [>10⁴ focus forming units (ffu)/pmolDNA) following transfection into NIH 3T3 cells. The FGFR2/BEK geneencodes two receptor species, FGFR2 and KGFR, with respect toligand-binding specificity (FIG. 1).

It was previously shown that KGFR transforms NIH 3T3 cells by creationof an autocrine loop whereas FGFR2 does not. KGF, unlike FGFR2, issecreted from NIH 3T3 cells and binds and activates KGFR (Miki et al.,Science 251:72-75, 1991). However, sequence analysis of theextracellular domain of the receptor revealed that it contained thethird Ig domain sequence of FGFR2. Other regions of the extracellulardomain were completely identical to the previously reported rat KGFR(Takagi et al., J. Biol. Chem. 269:23743-23749, 1994). Activatingmutations in transmembrane domains of EGF receptor and FGFR3 werereported in rat leukemia and human achondroplasia, respectively(Ben-Levy, J. Biol. Chem. 267:17304-17313, 1992). However, thetransmembrane domain of FGFR2 from ROS cells is also identical to ratKGFR, indicating that neither the extracellular or transmembrane domainof FGFR2 from ROS cells contains sequences that are known to activatethe receptor function. Therefore, we reasoned that the FGFR2 from ROScells, designated FGFR2-ROS, has been activated by an unknown mechanismand further examined the structure and function of the receptor.

FGFR2-ROS contains an altered C-terminus. Sequence analysis of theentire FGFR2-ROS cDNA predicted an isoform of FGFR2 with two Ig domainsand an acidic region (FIG. 1). Comparison of FGFR2-ROS with mouse KGFRand FGFR2 showed that it encodes an FGFR2 variant that diverges fromFGFR2 at amino acid 763. Following amino acid 763, FGFR2-ROS contains aunique 313 amino-acid stretch at its C-terminus (FIG. 1). No matcheswere observed when protein data bases were searched for homology toknown sequences with the unique C-terminal region of FGFR2-ROS. Inaddition, the nucleotide sequence encoding the unique C-terminus and 290bp of flanking 3′-untranslated sequence of the FGFR2-ROS cDNA revealedlittle identity to any known DNA sequences, suggesting that the 3′portion of FGFR2-ROS cDNA is derived from an unknown gene.

FGFR2-ROS is fully activated in a ligand-independent manner. To examinethe regulatory effect of the C-terminal domain, we utilized a naturalisoform of mouse KGFR, KGFR-ET (ET refers to “early termination”), thatcontained a partially truncated C-terminus that is identical to theC-terminus of the human FGFR2 isoform, TK25 (Chanpion-Arnaud et al.,Oncogene 6:979-987, 1991). To avoid receptor activation by KGF secretedfrom NIH 3T3 cells, the extracellular domain of KGFR and KGFR-ET wasreplaced with the extracellular domain of FGFR2-ROS, thereby generatingFGFR2-WT and FGFR2-ET, respectively (FIG. 1). A comparison of theC-terminal sequences of FGFR2-WT (SEQ ID NO: 1), FGFR2-ROS (SEQ ID NO:3) and FGFR2-ET (SEQ ID NO: 2) is shown in FIG. 2.

FGFR2-WT, FGFR2-ET and FGFR2-ROS were compared for their ability tomorphologically transform NIH 3T3 cells. Only FGFR2-ROS exhibitedhigh-titer transforming activity (Table 2). The activity was even higherthan that of KGFR, which can be bound and activated by KGF secreted fromthe host cells. Lack of significant transforming activity in FGFR2-WTindicates that the ligand-binding domain of FGFR2-ROS is functional andcan suppress the catalytic domain of the normal receptor, since theFGFR2-WT clone is a chimera of the ligand-binding domain of FGFR2-ROSand the cytoplasmic domain of KGFR (FIG. 1). Therefore, FGFR2-ROSappeared to have been maximally activated in a ligand-independentmanner. On the other hand, FGFR2-ET, an FGFR2 isoform with a differentC-terminal alteration, was weakly transforming (Table 2). The fociinduced by FGFR2-ET transfection were less aggressive than those ofFGFR2-ROS and could not be identified within 10 days after transfection,whereas FGFR2-ROS-induced foci were much more aggressive and wereclearly detectable earlier in the course of the transfection. Incontract, FGFR2-WT did not show significant transforming activity evenat this later stage. These data suggest that partial deletion orreplacement of the C-terminus of the FGFR2 can mediate ligandindependent activation.

Growth in semi-solid medium of NIH 3T3 transfectants expressing thesereceptor was also examined. Transfectants containing only vector orFGFR2-WT did not grow efficiently, whereas FGFR2-ET and KGFR showedmodest cloning efficiency (Table 2). In contrast, FGFR2-ROS showedmarkedly higher efficiency in this assay, indicating the highlyactivated state of this receptor. Nonetheless, all of the NIH 3T3 cellstransfected with FGFR2 expression vectors induced tumors efficientlywhen these transfectants were injected into athymic nude mice. Thisresult suggests that overproduction of FGFR2 generated tumors underthese conditions, since the ligands of FGFR2, such as αFGF, may beavailable for receptor activation in nude mice.

TABLE 2 Oncogenic Activity of FGFR2 Variants Transforming Cloning DNAActivity* efficiency† Tumorigenicity⇓ pCEV27  <1 × 10⁰ 0.2 0/6 KGFR 1.4× 10⁴ 2.7 6/6 FGFR2-WT  <1 × 10⁰ .03 6/6 FGFR2-ET 8.0 × 10² 1.8 6/6FGFR2-ROS 2.6 × 10⁴ 20.5 6/6 *NIH 3T3 fibroblasts were transfected withserial dilutions of the indicated plasmid DNAs plus 40 μg of calf thymusDNA as carrier. The number of foci produced by each DNA were countedafter three weeks and transformed activity was expressed as the numberof ffu per pmol DNA (ffu/pmol, n = 4). †To estimate cloning efficiency,G418-selected transfectants were suspended in 0.4% soft agar in thepresence of DMEM containing 10% calf serum and the number of colonieswere counted after 23 days in culture. ⇓1 × 10⁵ or 1 × 10⁶ NIH 3T3 cellstransfected with the indicated plasmids were injected subcutaneouslyinto athymic mice and tumors were scored 18 days following injection.

The altered C-terminal tail of FGFR2-ROS is required for fullactivation. Comparison of the C-termini of FGFR2-ET and FGFR2-ROS withthat of FGFR2-WT (SEQ ID NO: 2, 3 and 1, respectively) suggested thatthe loss of the C-terminal domain may be responsible for the highertransforming activity of the variant FGFRs. To examine if the C-terminaltail of FGFR2-ROS has an important role in receptor activation, thissequence was deleted from FGFR2-ROS to generate FGFR2Δ764-. Thetransforming activities of Δ764 and FGFR2-ROS were then compared aftertransfection of NIH 3T3 cells. Whereas FGFR2-ROS exhibited a hightransforming activity even at an early stage (2.6×10⁴ ffu/pmol DNA),Δ764- did not show significant transforming activity. However, Δ764-showed weaker transforming activity (5.0×10² ffu/pmol DNA) at a laterstage. FGFR2-ROS was 50-fold more active than Δ764- at this later stage.These results strongly indicate that the C-terminal sequence ofFGFR2-ROS plays a major role in the activation of this receptor variant.

FGFR2-ROS is highly phosphorylated in NIH 3T3 transfectants. To assessthe effects of different C-terminal sequences on receptorphosphorylation, FGFR2 was immunoprecipitated from soluble lysates (2mg) of pCEV27- or FGFR2-NIH 373 transfectants with an affinity purifiedanti-FGFR2 antiserum (αFGFR2). Immunoprecipitated FGFR2 was thenanalyzed for phosphorylation levels by SDS-polyacrylamide gelelectrophoresis (PAGE, 8%), transfer of the immunoprecipitated proteinsto Immobilon-P, and blotting with either an anti-phosphotyrosineantibody (αPTYR) or αFGFR2). The wild-type receptor displayed a lowlevel of receptor phosphorylation, whereas phosphorylation of FGFR2-ETand Δ764- proteins was detectable only after a longer exposure of theautoradiogram. In contrast, FGFR2-ROS was identified as a broad band ofgreater than 300 kDa and phosphorylated at a similar level as KGFR,which was activated by KGF secreted from the transfectants. A minor band(180 kDa) of FGFR2-ROS was barely detectable with αFGFR2 antibody butreadily detectable with αPTYR, indicating that this minor band was alsohighly phosphorylated. Immunoblot analysis of the sameimmunoprecipitates with αFGFR2 antibody revealed that the expressionlevels of different FGFR2 constructs in the NIH 3T3 transfectants werecomparable. FGFR2-WT, FGFR2-ET and Δ764- were identified as broad bandsof approximately 160 kDa as well as lesser abundant 110 kDa, 100 kDa and90 kDa bands, respectively. The receptor in the KGFR transfectant wasidentified as 110 kDa and 95 kDa forms. The larger broad bands observedin these transfectants probably represent receptor species modified bypost-transnational modifications of the extracellular domains byglycosaminoglycans, since treatment with enzymes that specificallydegrade glycosaminoglycans reduced the size of these receptors to lowermolecular weight forms that roughly correspond to their predictedmolecular sizes.

FGFR2-ROS appears to form unusually stable dimers. The cDNA encodingFGFR2-ROS predicts a protein with a molecular mass of 110 kDa.However, >300 kDa and 180 kDa species were observed as the predominantand minor forms of the receptor in FGFR2-ROS transfectants,respectively. To further analyze the FGFR2 variants, NIH 3T3 cellsexpressing the indicated constructs were incubated in the presence (+)or absence (−) of disuccinimidyl suberate for 20 minutes at 4° C.,lysed, and immunoprecipitated with αFGFR2. The immunoprecipitatedsamples were subjected to SDS-PAGE (6%) and blotted with αFGFR2antibodies. The molecular mass of the major form of FGFR2-ROS wasapproximately 360 kDa, suggesting that this form represents dimers ofthe minor form (approximately 180 kDa). The minor broad band wasconverted to a discrete band of 110 kDa by enzymes which digest sugarmoieties. Therefore, the minor form may represent the modified monomerof FGFR2-ROS.

Chemical crosslinking was performed on NIH 3T3 transfectants expressingKGFR, which can been activated by KGF secreted by NIH 3T3 cells. A largefraction of KGFR protein was detected as receptor dimers followingcrosslinker exposure. In the absence of the crosslinker, the majority ofKGFR protein was detected in its monomeric form. In contrast, a muchlower level of receptor dimers was detected in the case of FGFR2-WT,which cannot be activated by secreted KGF. Interestingly, the mobilityof receptor protein in FGFR2-ROS transfectants was unaltered in bothcrosslinker-treated and untreated cells, suggesting that most of thereceptor had already been dimerized. These dimers were very stable,since the larger receptor species were detected in SDS-PAGE underreduced and denatured conditions and were highly phosphorylated ontyrosine. These results may indicate that the C-terminal-encodedsequence of FGFR2-ROS mediates usually stable receptor dimer formationin the absence of ligand. Constitutive dimerization andautophosphorylation may therefore underlie the potent transformingactivity of FGFR2-ROS.

The FGFR2 gene is rearranged in ROS cells. To examine if the alteredC-terminal domain of FGFR2-ROS was generated by chromosomalrearrangement of the FGFR2 gene in the osteosarcoma cell line, normalrat kidney (NRK) or ROS cell genomic DNA was analyzed by Southernblotting. NRK or ROS cell genomic DNA was digested with BglII, BamHI orSpeI, separated on a 0.7% agarose gel, and transferred to a nylonmembrane. The blot was hybridized with a 0.38-kb BglII-HindIII fragmentof the FGFR2-WT cDNA that is derived from the 3′ one-third of the codingsequence of FGFR2-WT cDNA (amino acids 697-822), which contains regionsthat are present and regions that are absent in FGFR2-ROS. The probedetected multiple DNA fragments in BglII (8 kb, 4 kb, 3.5 kb), BamHI (10kb, 7 kb), and SpeI (10.5 kb, 7.5 kb) digests of normal genomic DNA. Incontrast, some of these fragments (BglII, 8 kb; BamHI, 7 kb; SpeI, 7.5kb) were not detected by the same probe in ROS cell genomic DNA,indicating that part of the region detectable by the probe is notpresent in ROS cell DNA.

Southern blot analysis was also performed using a probe from a regionthat spans the FGFR2-FRAG1 junction (amino acids 697-764 of FGFR2 and 70amino acids derived form the fusion partner gene). ROS cell DNAcontained several DNA fragments that were not detected in normal cells(e.g., BglII 4 kb, BamHI 8 kb and 6 kb, SpeI 8 kb and 4 kb). Therefore,ROS cells contain DNA fragments that are not present in normal DNA.These results strongly suggest that the FGFR2 gene has undergone astructural rearrangement in ROS cells that occurs in the region encodingthe C-terminal domain of the receptor.

Cloning and structure of the fusion partner gene, FRAG1. The experimentsdescribed above established that FGFR2-ROS was generated by a genefusion between FGFR2 and a novel gene, which we designate rat FRAG1 (SEQID NO: 5). Since acquisition of the FRAG1 sequence played a criticalrole in FGFR2 activation in ROS cells, we isolated cDNA for thewild-type rat FRAG1 gene. A probe derived from the FRAG1 region ofFGFR-ROS cDNA was used to screen a rat brain cDNA library. A plasmidwith the largest cDNA insert, FRAG1 CL26 (1.6 kb), was chosen forcharacterization. Since the detection of a 2.0 kb FRAG1 mRNA by northernblot analysis (see below) suggested that the rat FRAG1 clones isolatedby library screening have been truncated, additional 5′ sequence wasobtained by anchored PCR (Frohman et al., Proc. Natl. Acad Sci. USA85:8998-9002, 1988). These combined cDNA sequences (1780 bp, SEQ ID NO:5) together constitute a nearly full-length rat FRAG1 cDNA (FIG. 3).Analysis of the rat FRAG1 cDNA revealed an open reading frame of 254amino acids, starting from an ATG codon located at positions 723-725,that encodes a protein with a predicted mass of 28 kDa (FIG. 4, SEQ IDNO: 6). An in-frame termination codon is located at positions 334-336.Therefore, the longest open frame starts at 129 amino acids upstreamfrom the first ATG, if any amino acid except methionine is considered asa start codon. Comparison of the FGFR2-ROS and FRAG1 sequences revealedthat the breakpoint was located 70 residues amino-terminal from thefirst ATG, and that the chromosomal rearrangement resulted in anin-frame fusion of both genes. These results indicate that the entirecoding sequence of FRAG1 has been fused with FGFR2 in ROS cells toactivate the receptor.

Nucleotide and protein data base searches revealed that rat FRAG1 wasnot identical to any known sequences. FRAG1 gene expression in rattissues was studied by northern blotting. A blot containing poly(A)⁺ RNA(2 μg) from various adult rat tissues was hybridized to a rat FRAG1 cDNAprobe. A 2.0 kb rat FRAG1 mRNA was observed in heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis. In addition, 1.3 kband 0.9 kb mRNA species were detected by the rat FRAG1 probe in testisand, to a lesser extent in lung. A 3.2 kb mRNA was also detected inlung, brain, spleen, kidney, and testis. Since FRAG1 is not related toany proteins with known functions, it was not possible to presume itsfunction on a structural basis. However, two unknown proteins thatshowed similarity to FRAG1 were found in protein data bases. Theseproteins, a 30 kDa protein of the nematode Caenorhabditis elegans(TO4A8.12, GenPept accession number Z35663) and a 107.9 kDa yeastprotein (SC108 kD, SwissProt accession number P25618), were deduced fromtranscription units identified in nematode and yeast genome sequencingprojects, respectively, indicating that FRAG1-related genes are wellconserved from mammalian to lower eukaryotic cells. FIG. 5 shows analignment of the 5′ ends of rat FRAG1 and corresponding sequences in the30 kDa nematode and 107.9 kDa yeast genes showing a well-conservedregion that is useful, for example, as a hybridization probe. FIG. 6shows an alignment of the deduced protein sequences of rat FRAG1 and the30 kDa nematode and 107.9 kDa yeast genes.

Subcellular localization of rat FRAG1. Altered subcellular localizationof receptors can result in their activation (Mitra et al., Proc. Natl.Acad. Sci. USA 84:6707-6711, 1987). Therefore, immunofluorescencestaining of FGFR2-WT and FGFR2-ROS transfectants using αFGFR2 antibodywas performed. Both of the receptor species were highly expressed in allsubcellular compartments, but expression of FGFR2-ROS in the cytoplasmwas slightly less abundant than FGFR2-WT. To examine whether the FRAG1sequence in FGFR2-ROS affects the subcellular localization of thereceptor, immunofluorescence staining of NIH 3T3 cells expressingepitope-tagged FRAG1 was performed. Little staining was observed in NIH3T3 cells transfected with vector alone (pCEV29F3), indicating a lowbackground of this system. In contrast, a strong signal was observedwhen immunofluorescence staining was performed on F3-FRAG1transfectants. In a large fraction of these cells, F3-tagged FRAG1showed a perinuclear staining pattern consistent with a localization inthe Golgi complex. In addition to this perinuclear localization, asubpopulation of cells also exhibited cytoplasmic staining, suggestingthat FRAG1 is translocated to the cytoplasm. These results may indicatethat the presence of FRAG1 in FGFR2-ROS affects its subcellularlocalization, which may contribute to the highly activated state ofFGFR2-ROS. The identification of FRAG1-like sequences in other species,ubiquitous expression of FRAG1 in adult tissues and perinuclearlocalization of FRAG1 protein suggest an important role for this gene incellular functions.

Example 2 Subcellular Localization of FGFR2-ROS (FGFR2-FRAG1)

Immununofluorescent staining of epitope-tagged rat FRAG1 proteinrevealed a discrete perinuclear subcellular localization. We reasonedthat the presence of the FRAG1 sequence on the FGFR2-ROS variant couldalter the location of the receptor. When the same experiments wereperformed on cells transfected with the FGFR2-ROS and FGFR2-WTreceptors, we found that the FGFR2-ROS receptor showed a perinuclearstaining similar to FRAG1 while the wild-type receptor exhibited astaining consistent with staining on the cell surface.

These results indicate that the presence of the FRAG1 sequence on theFGFR2 alters the subcellular localization of the receptor, which mayunderlie the potent transforming activity of this receptor variant.

Example 3 Preparation of Anti-FRAG1 Antibodies

Two peptides derived from the predicted protein sequence of rat FRAG1were used to generate antisera against FRAG1 protein. These peptideswere synthesized as MAP peptides which consist of a core residue fromwhich four arms of identical peptides are produced. The generation ofsuch peptides obviates the need to couple the peptides to a carrierprotein to enhance antigenicity. The resulting peptides, N-FRAG1(residues 1-16 of SEQ ID NO: 6) and C-FRAG1 (residues 240-254 of SEQ IDNO: 6), were injected into rabbits to generate anti-FRAG1 antibodies.These antisera recognized a 28 kDa protein in both untransfected(endogenous) and FRAG1-transfected (overexpressing) cells.

Example 4 Isolation of Human FRAG1

We have isolated the human homolog of rat FRAG1. A cDNA library preparedfrom human fibroblast cells, M426, was screened with a rat FRAG1 cDNAprobe. Positive plaques were rescreened two additional times to ensurethe identity and purity of the clones. Five positive clones wereidentified with inserts in the ranging from 1.5 kb to 1.8 kb. InternalDNA sequence revealed that these cDNAs encoded the human homolog of ratFRAG1. The cDNA containing the largest insert, hFRAG1-5CA, was chosen todetermine the entire DNA sequence.

Over 90% of the human FRAG1 sequence has now been determined (FIG. 7,SEQ ID NO: 11). A search of nucleotide sequence databases revealed thathuman FRAG1 is highly related to the rat FRAG1 seauence but containsunique regions not present in the rat FRAG1 sequence. The size of thehuman FRAG1 mRNA on Northern blots suggest that the human FRAG1-5CAclone is full length.

Example 5 Isolation of Genomic Clones and Homologs of Rat and HumanFRAG1

The skilled artisan can obtain the genomic clones for rat and humanFRAG1 by screening genomic libraries with the respective cDNA sequences(or fragments thereof) as probes or primers under high stringencyhybridization conditions using conventional nucleic acid hybridizationtechniques.

In order to obtain a FRAG1 homolog from a species other than rat orhuman, a cDNA (or genomic library) is screened with (1) a nucleic acidprobe or primer having complete sequence similarity with either the rator human FRAG1 cDNA sequence or both (including the respectivefull-length cDNA(s)); a degenerate nucleic acid probe from a region thatis highly conserved between rat and human FRAG1; for expressionlibraries, an antibody probe that specifically recognizes either rat orhuman FRAG1 (and preferably both rat and human FRAG1). Appropriate cDNAand genomic libraries, including expression libraries, are widelyavailable and are commercially available from a variety of sources.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

1. A method of detecting a chromosomal rearrangement consisting offusion of a fibroblast growth factor receptor activating gene 1 (FRAG1)gene to a fibroblast growth factor receptor 2 (FGFR2) gene in achromosome of a subject, comprising: incubating a chromosome of asubject with a nucleic acid molecule under conditions that allow thenucleic acid molecule to hybridize specifically with the chromosome,wherein the nucleic acid molecule comprises the complement of a nucleicacid sequence encoding amino acids 697-822 of FGFR2-WT and hybridizesunder stringent conditions to a FRAG1 nucleic acid sequence comprisingSEQ ID NO: 11, wherein the stringent conditions consist of hybridizationat 65° C. in a hybridization solution comprising 6× SSC, 5× Denhardt'sreagent, 0.5% SDS, 100 μg/ml denatured and fragmented salmon sperm DNA;and comparing hybridization of the nucleic acid molecule to thechromosome with hybridization of the nucleic acid molecule to a controlchromosome lacking the chromosomal rearrangement, thereby detecting thechromosomal rearrangement.
 2. The method of claim 1, wherein the nucleicacid molecule is a primer.
 3. The method of claim 1, wherein the nucleicacid molecule is a probe.
 4. The method of claim 3, wherein the probe isattached to a detectable label.
 5. The method of claim 4, wherein thedetectable label is a radioactive isotope, a ligand, a chemiluminescentagent, an enzyme, or a combination thereof.
 6. The method of claim 1,wherein the subject has an osteosarcoma.
 7. The method of claim 1,wherein the chromosomal rearrangement is diagnostic for osteosarcoma. 8.The method of claim 1, wherein incubating comprises performingfluorescence in situ hybridization.
 9. The method of claim 1, whereinincubating comprises performing nucleic acid amplification.
 10. A methodof detecting a chromosomal rearrangement consisting of fusion of afibroblast growth factor receptor activating gene 1 (FRAG1) gene to afibroblast growth factor receptor 2 (FGFR2) gene in a chromosome of asubject, comprising: incubating a chromosome of a subject with a nucleicacid molecule under conditions that allow the nucleic acid molecule tohybridize specifically with the chromosome, wherein the nucleic acidmolecule comprises the complement of a nucleic acid sequence encodingamino acids 697-822 of FGFR2-WT and hybridizes under stringentconditions to a FRAG1 nucleic acid sequence that encodes a polypeptidecomprising SEQ ID NO: 12, wherein the stringent conditions consist ofhybridization at 65° C. in a hybridization solution comprising 6× SSC,5× Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured and fragmentedsalmon sperm DNA; and comparing hybridization of the nucleic acidmolecule to the chromosome with hybridization of the nucleic acidmolecule to a control chromosome lacking the chromosomal rearrangement,thereby detecting the chromosomal rearrangement.
 11. The method of claim10, wherein the nucleic acid molecule is a primer.
 12. The method ofclaim 10, wherein the nucleic acid molecule is a probe.
 13. The methodof claim 12, wherein the probe is attached to a detectable label. 14.The method of claim 13, wherein the detectable label is a radioactiveisotope, a ligand, a chemiluminescent agent, an enzyme, or a combinationthereof.
 15. The method of claim 10, wherein the subject has anosteosarcoma.
 16. The method of claim 10, wherein the chromosomalrearrangement is diagnostic for osteosarcoma.
 17. The method of claim10, wherein incubating comprises performing fluorescence in situhybridization.
 18. The method of claim 10, wherein incubating comprisesperforming nucleic acid amplification.
 19. A method of detecting achromosomal rearrangement consisting of fusion of a fibroblast growthfactor receptor activating gene 1 (FRAG1) gene to a fibroblast growthfactor receptor 2 (FGFR2) gene in a chromosome of a subject, comprising:incubating a chromosome of a subject with a nucleic acid molecule underconditions that allow the nucleic acid molecule to hybridizespecifically with the chromosome, wherein the nucleic acid moleculecomprises the complement of nucleotides 546-569 of SEQ ID NO: 5 whereinthe conditions consist of hybridization at 65° C. in a hybridizationsolution comprising 6× SSC, 5× Denhardt's reagent, 0.5% SDS, 100 μg/mldenatured and fragmented salmon sperm DNA; and comparing hybridizationof the nucleic acid molecule to the chromosome with hybridization of thenucleic acid molecule to a control chromosome lacking the chromosomalrearrangement, thereby detecting the chromosomal rearrangement.
 20. Amethod of detecting a chromosomal rearrangement consisting of fusion ofa fibroblast growth factor receptor activating gene 1 (FRAG1) gene to afibroblast growth factor receptor 2 (FGFR2) gene in a chromosome of asubject, comprising: incubating a chromosome of a subject with a nucleicacid molecule under conditions that allow the nucleic acid molecule tohybridize specifically with the chromosome, wherein the nucleic acidmolecule comprises the complement of a nucleic acid sequence encodingamino acids 1-13of SEQ ID NO: 3, wherein the conditions consist ofhybridization at 65° C. in a hybridization solution comprising 6× SSC,5× Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured and fragmentedsalmon sperm DNA; and comparing hybridization of the nucleic acidmolecule to the chromosome with hybridization of the nucleic acidmolecule to a control chromosome lacking the chromosomal rearrangement,thereby detecting the chromosomal rearrangement.